RESEARCH ARTICLE Open Access

Significance of Cuscutain, a cysteine proteasefrom Cuscuta reflexa, in host-parasite interactionsMarc Bleischwitz1, Markus Albert2, Hans-Lothar Fuchsbauer3, Ralf Kaldenhoff4*

Abstract

Background: Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well asbiochemical changes in the host and the parasite. These modifications could be caused by a change in protein orgene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the hostattachment site were identified.

Results: One of the infestation specific Cuscuta genes encodes a cysteine protease. The protein and its intrinsicinhibitory peptide were heterologously expressed, purified and biochemically characterized. The haustoria specificenzyme was named cuscutain in accordance with similar proteins from other plants, e.g. papaya. The role ofcuscutain and its inhibitor during the host parasite interaction was studied by external application of an inhibitorsuspension, which induced a significant reduction of successful infection events.

Conclusions: The study provides new information about molecular events during the parasitic plant - hostinteraction. Inhibition of cuscutain cysteine proteinase could provide means for antagonizing parasitic plants.

BackgroundParasitic weeds such as Cuscuta reflexa are obligateholoparasites with low host specificity. The plants arefound in areas with relatively mild climates around theworld. In farming regions, these parasites cause substan-tial damage to many commercially important crops suchas sugar beet, alfalfa, pepper, cucumber, tomato potatoor allium [1]. Currently, an effective control of Cuscutaoutbreaks is based on preventive strategies includingcontrol of seed contamination and application of herbi-cides prior to seed emergence. The use of herbicides oninfected plants with an established host parasite interac-tion only appears to be successful and not harmful tothe host plant if the host is herbicide resistant [2,3]. Dueto difficulties with conventional breeding techniques,molecular biology genomic research on parasites isneeded to develop new control strategies [4-8]. Researchon host reactions to parasitic plant infection in modelplants such as Arabidopsis thaliana, Medicago trunca-tula and crops like tomato or tobacco have already gen-erated promising results [9-12].

In Cuscuta spp. photosynthesis is reduced or absent[13]. Consequently, the plant depends on carbohydrateswithdrawn from the host plant [14]. A connection (haus-torium) at the contact site is established through thesecretion of enzymes and sticky substances consistingmainly of de-esterified pectins [15]. At early stages ofCuscuta invasion, host plants react with specific geneexpression to regulate processes including calciumrelease, cell elongation and cell wall modification (Albert,Werner, Proksch, Fry, & Kaldenhoff 2004; Werner, Ueh-lein, Proksch, & Kaldenhoff 2001; [16]. A gene coding foran arabinogalactan protein (AGP) was found to be up-regulated in tomato at an early stage of infection and ithas a significant function for C. reflexa attachment to thehost plant (Albert, Belastegui-Macadam, & Kaldenhoff2006). After attachment, the host is invaded by hyphaeand chimeric cell walls of host and Cuscuta cells areformed [17]. Phloem and xylem connections transferwater, nitrogen-compounds, assimilates and even RNA,proteins or plant viruses from the host to the parasiticplant [18-20].The current knowledge about gene expression in the

parasite Cuscuta at early stages of infection is limited.Besides host responses, parasitic plant reactions need tobe determined for a complete elucidation of the

* Correspondence: kaldenhoff@bio.tu-darmstadt.de4Darmstadt University of Technology, Applied Plant Science, Schnittspahnstr.10, 64287 Darmstadt, GermanyFull list of author information is available at the end of the article

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© 2010 Bleischwitz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

infection process. This knowledge will likely be one ofthe prerequisites for the improvement of strategies toprevent or control Cuscuta-infection. For a first over-view of parasite responses, we have constructed a Cus-cuta cDNA-library corresponding to mRNAs specific forearly stages of haustoria development. Here, we describeone of the identified genes, which encodes a Cuscutareflexa haustoria specific cysteine protease that wenamed cuscutain. Its expression, biochemical character-istics and significance during the infection processopens the possibility to develop a cuscutain-based strat-egy against Cuscuta infection.

ResultsCuscutainmRNA from Cuscuta tissue containing haustoria wasemployed to construct a cDNA library with 7000 primarytransformants. Putative haustorium specific expression ofthe corresponding genes was identified by differentialcDNA hybridization (haustorium containing materialversus shoot without haustoria) to all obtained cDNAclones assigned on a macroarray. 16 different clones werepreselected by this procedure showing a more intensesignal on a macroarray chip when hybridized with acDNA probe from RNA of haustoria containing tissue.One of the signals with a remarkable differential intensitycorresponded to a cDNA clone derived from a cysteineproteinase encoding mRNA (Accession No.: FB701665).Because cysteine proteinases were known to participatein some interspecies interactions, we were interested tostudy the role of this Cuscuta reflexa enzyme. For verifi-cation of spatial expression, Cuscuta-RNA from the twotissue types was further characterized by Northern blotwith a probe derived from the above said cDNA clone.Just a faint signal was obtained in RNA from Cuscutashoots lacking haustorial structures and a strong one wasobtained in RNA from shoot material with haustoria,which was harvested at three days post attachment(Figure 1). Sequence analysis of the deduced mRNArevealed that translational start and stop codons includ-ing poly A tail were cloned as cDNA indicating a fulllength cDNA-insertion. Sequence comparison obtainedfrom BLAST-n at NCBI revealed a high sequence identityto cysteine proteinases like Ipomoea batatas papain-likecysteine proteinase isoform II (86%), Phaseolus vulgarisMoldavian encoding cysteine proteinase (78%) or Arachishypogaea cysteine protease-like protein (77%). An equallyhigh identity to numerous cysteine proteinases wasobserved when the translated sequence was subjected toa comparison with entries from Expasy Proteomics Ser-ver at Swiss Institute of Bioinformatics (e.g. 86% overallidentity to Ipomoea batatas papain-like cysteine protei-nase isoform II). According to sequence alignment andprotein domain identification tools, specific functional

sites could be identified (Figure 2). Hence, the predictedprotein consists of a prepeptide, thought to be responsi-ble for its extracellular localization (98.2% probability).The corresponding cleavage site between prepeptide andthe subsequent propeptide is represented by a SSSDDsequence (Figure 2, 99.9% probability). Next to the pre-peptide from N- to C-terminus the propeptide harbors aso called ERFIN-motif [21], which is a stabilizing compo-nent of intramolecular interaction. This protein regionwas described to form an inhibitor of the proteinaseactivity [22]. Furthermore the propeptide acts as an intra-molecular chaperone [23]. The actual cysteine proteinaseregion contains characteristic active sites as indicated bymotiv search (Prosite, Swiss Institute of Bioinformatics)and comprises a protein with a calculated molecularweight of 24.8 kDa. That of the prepeptide is 11.7 kDa.

Biochemical characterization of cuscutain and theinhibitory propeptideThe coding sequence for the propeptide inhibitor regionand that for the enzymatic activity were separatelycloned into an E. coli expression vector. After induction,both proteins were expressed in E. coli and could beidentified in crude extracts of soluble proteins by SDSgel electrophoresis and Coomassie staining. Nickel-column-chromatography was employed to purify theC-terminal 6xHis tagged proteins and provided a pro-tein solution without significant impurities in the caseof the propeptide as demonstrated by SDS gel electro-phoresis and Coomassie staining (Figure 3). For the

shoot haustoria

cuscutain

Figure 1 Northern blot with total Cuscuta RNA. Cuscuta RNAfrom shoots (shoot) or shoot material with haustoria (haustoria).Upper panel: hybridization signal with a cuscutain-cDNA probe.Lower panel: Ethidium bromide stained total RNA to indicate evenloading.

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enzymatic protein portion a further purification stepafter gel-electrophoresis was applied as outlined. Even-tually, also the enzymatic protein part was processed topurity as indicated by the staining of the correspondinggel (Figure 3). The purified enzymatic protein compo-nent was subjected to enzymatic characterization andthe putative inhibitor component was tested for inhibi-tor efficiency. The results are summarized in Figure 4and revealed for the Cuscuta cysteine proteinase a Km =0,393 ± 0,02 mM or a turnover number of 3.5 anilinebonds/s (Figure 4A), an optimum at pH 7.0- 7.5 (Figure4B) and a temperature optimum at 40°C (Figure 4C).These figures are in a comparable range to othercysteine proteinases (Table 1). In accordance to thenaming of the papaya cysteine proteinase papain, theCuscuta enzyme was denominated cuscutain. For thepredicted inhibitory propeptide a Ki of 0.7 ± 0.02 nMwas determined on cuscutain activity (Fig 4D). The inhi-bitory effect on other cysteine proteinases like papain orcruzipain was found to be 20 times lower indicating acuscutain-specific inhibitory function. The data supportthe sequence predictions concerning the biochemicalfunction for both the enzyme and the inhibitorcomponent.

Biological function of cuscutainSince cuscutain-mRNA was abundant in haustoria, afunction of the encoded proteinase for the infection pro-cess was assumed. If cuscutain activity is essential for asuccessful infection, inhibition of the enzyme could beone way of reducing the effectiveness or preventing Cus-cuta infestation. To test this assumption, 60 tobaccoplants were prepared for infection by curling 20 cmCuscuta shoot segments around the host shoot. Onehalf of the assay was sprayed with 100 μg/ml buffered

inhibitor propeptide solution 2 times a day for oneweek. As a mock control the other 30 plants were trea-ted with buffer solution only. The propeptide solutionhad no visual effect on host tobacco plants e.g. with

Figure 2 Predicted cuscutain sequence. Prepeptide (green), propeptide (red) and the catalytic protein (blue) are depicted as well as thecleavage site and the ERFIN-motif (black, underlined).

30 kDa

25 kDa

20 kDa

15 kDa

Figure 3 Protein separation . Left panel: Coomassie stainedproteins from E. coli prior to induction (control) or after a 2 hourinduction separated by SDS gel electrophoresis (enzyme: E. coliexpressing the catalytic protein; propeptide: E. coli expressing thepropeptide). Right panels: purified cuscutain (enzyme) orpropeptide.

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regard to development. On a daily basis, the plants weremonitored for prehaustoria, haustoria and attachedhaustoria. Parasites on inhibitor propeptide solutiontreated Cuscuta - host plants appeared to be thinnerand less vital as compared to controls (Figure 5A, B).For a quantitative assessment, the number of prehaus-toria and haustoria attached or not attached to the host,were counted and related to Cuscuta shoot length.Untreated plants developed on average 9 (± 2.1) haus-toria per 10 cm, 6 (± 1) of these produced a successfulconnection via hyphae. Inhibitor treated plants had 5.6(± 1.6) haustoria per 10 cm with 1.5 (± 0.5) successfulpenetrations and connections to the host vascular tissue.

On average treatment with the inhibitory propeptidesolution reduced haustoria formation roughly by 40%and decreased successful parasite infestation from 65%down to 15%. In most cases (29 out of 30), Cuscuta ontreated plants dried out after about two weeks withoutfurther spraying (Figure 5c, d), which suggests that aminimum number of successful connections to hostxylem and phloem per parasite shoot length is requiredfor vitality and propagation of C. reflexa. It also indi-cates that an active cuscutain cysteine proteinase is acomponent of a successful Cuscuta infestation.

DiscussionA role of cuscutain during the infection process is sug-gested by the presented data. Accordingly, the sequenceof related molecular events could be envisaged as follows:The cuscutain gene is activated concomitant to haustoriaformation. The gene encodes a so called pre-pro-protein,with each of the protein subunits having a separate func-tion. The prepeptide targets the cuscutain primary pro-tein to the extracellular space. Here the unprocessedtranslation product is cleaved and deleted from the pre-

Figure 4 Biochemical characterization of cuscutain. A Hanes-Woolf plot of cuscutain activity for Km estimation. B relative cuscutain activity atdifferent pH conditions. C relative cuscutain activity at different temperatures. D Dixon plot of cuscutain activity for estimation of inhibitor Ki.

Table 1 Biochemical characteristics of cysteineproteinases

Cuscutain Cruzipain Papain

temperature Optimum [°C] 40 37 25

pH 7,5 8,0 6,5

Km [mM] 0,4 0,003 0,4

Biochemical characteristics of cuscutain, cruzipain [49] and papain [50,51].

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and propeptide. Deletion of the inhibitor-propeptide con-verts cuscutain from an inactive form to an activeenzyme with a cysteine proteinase function. Outside theparasite, the enzyme fulfills a role in the successful infec-tion process, possibly by weakening host structuresthrough protein degradation. Therefore, addition ofwater inhibitor solution by spraying most likely restrictsthis enzymatic activity outside the haustorial cells. It isyet unclear how a host-specific cuscutain activity isachieved, since the enzyme is most likely localized in thevicinity of host and Cuscuta tissue. If a concentrationgradient of effective inhibitor components from parasiteto host is created from primary cuscutain processing, theenzyme would show higher activity close to the host. Pro-tective structures, e.g. the high degree of pectins on haus-toria surfaces, could be another factor favoring thedegradation of host cells in comparison to parasite tissue.It is assumed that degrading enzymes, which are either

parasite- or host-encoded support the penetration ofparasitic hyphae [24,25]. In this regard the role of cuscu-tain resembles that of Orobanche encoded enzymes

which were located in the cytoplasm and cell walls ofintrusive cells and in the adjacent host apoplast duringhaustorium penetration [26-29].

ConclusionsPapain-like cysteine proteases have been identified at thesurface of various interaction surfaces between plantsand pathogens like bacteria, fungi, oomycetes, nematodesinsects or herbivores [30-40]. Some of these are a compo-nent of a defense mechanism while others are implicatedin the parasitic pathogenic attack [41]. The identificationof the cysteine proteinase cuscutain as a component thatmay be important for successful infestation of the parasi-tic plant C. reflexa could open the possibility for a newapproach for development of parasitic plant blockingagents. During the parasite - host interactions both plantspecies act and react in order to invade, prevent, or toler-ate invasion. Among others, these responses are visible asdifferential gene expression [42]. The identification of thecorresponding proteins increases our knowledge aboutthe molecular events of plant parasite infection. As

Figure 5 Cuscuta reflexa on tobacco. The parasite successfully attached to untreated plants (A, C), but not propeptide solution treatedplants (B, D).

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demonstrated, the encoded proteins could also be signifi-cant for the host parasite interaction. There is a chancethat a reduction of parasite-derived proteins weakens theparasite’s infection efficiency and thereby strengthenshost defense. However, prior to an application of a cuscu-tain propeptide solution in farming to protect crops someuncertainties must be ruled out. The inhibitor studiesshowed that its action spectrum is quite specific for cus-cutain. It is unknown how similar cysteine proteasesfrom other Cuscuta species or other parasitic plants areaffected or if the inhibitor is effective on related pro-teases. It is possible that only one inhibitor is effectiveper species. On the other hand, the activity of cysteineproteinases could play a role in other parasitic plantinteractions such as those with Orobanche or Striga.Although the latter parasitic weeds are root- and notshoot-parasites, the possibility of consistencies at themolecular level exists. Inhibition of cysteine proteasescould thus be of wider importance for antagonizing para-sitic plants from different genera.

MethodsPlant material and growth conditionsTobacco plants were grown in standard potting soilunder 16 h/8 h day/night light conditions (800 μmolphotons m2 s-1) at 25 °C. Cuscuta reflexa was grown onColeus blumei host plants under the same greenhouseconditions as the tobacco plants. Coleus blumei waschosen as host plant for Cuscuta cultivation because ittolerated this parasitic infection. C. reflexa was propa-gated vegetatively throughout. For infection of tobaccoplants, C. reflexa shoot tips of about 20 cm length werewrapped around a wooden stick. After 24 h, C. reflexashoots were transferred to 4-5-week-old tobacco plantsand curled around the stem; this time point was set asthe starting point of the infection process.

Northern blot analysisRNA isolation from Cuscuta shoots was performedusing the RNAeasy plant mini kit (Qiagen) following themanufacturer’s instructions. The gel was loaded with 5μg RNA per lane and blotted onto a nylon membrane(Applichem Inc., Darmstadt, Germany). The blot washybridized with a cDNA probe comprising 270 bp ofthe open reading frame encoding the Cuscutain-enzymeactive site. It was synthesized by PCR (forward primer:5’-GGCGCGCCCCATACATTTGCTCCAAGCGG-3’;reverse primer: 5’-ATTTAAATGTGCTAACAGCTGC-CACAGTTG-3’). The PCR reaction was carried outusing DIGlabelled dUTPs. Membrane blots were pre-hybridized for 2 h in DigeasyHyb (Roche DiagnosticsGmbH, Mannheim, Germany); subsequently, the dena-tured probe was added for overnight hybridization at 42°C. Washing and detection was performed following the

manufacturer’s protocol. Detection was carried out fol-lowing the suppliers protocol (Pharmacia Biotech,Munich, Germany) using an alkaline phosphatase-conjugated antibody and CDPstar as substrate for che-miluminescence reaction. The chemiluminescence wasvisualized and quantified using a chemiluminescencedetector equipped with a digital camera and quantifica-tion software (BioRad).

Comparative macroarrayTissue samples of Cuscuta reflexa including prehaustoriaand haustoria were collected 3 days after the infection of5 weeks old tobacco plants. Total RNA was isolatedusing RNAeasy plant mini kit (Qiagen, Germany) follow-ing the manufacturer’s instructions. 2 μg total RNA wasemployed for first strand cDNA synthesis (Ready-To-Go™

You-Prime First-Strand Beads; GE Healthcare)using 3’CDS-primer and SMARTIIA-oligo-primer fromSMART™ cDNA synthesis system (Clontech). cDNA wasPCR amplified applying the above mentioned primersand the product was cloned via TA cloning (TOPO TACloning®, Invitrogen). Colonies of transformed bacteriawere selected on Ampicillin (50 μg/ml) containing agarplates using blue/white selection (LB-agar plates: 1%NaCl, 1% Trypton, 0.5% yeast extract, 1.5% agar, 60 μg/ml Isopropyl-b-D-thiogalactopyranosid, 40 μg/ml X-Gal).White colonies were transferred into a 96 well plate, eachwell filled with 200 μl LB (1% NaCl, 1% Trypton, 0.5%yeast extract), grown overnight and stored after additionof one drop of glycerol (90%) at -80°C. An aliquot of theE.coli stock (10 μl) was subjected to PCR amplification ofthe plasmids cDNA insertion using vector specific pri-mers and following standard procedures to a product endconcentration of 70- 100 ng/μl in a 96 well plate. Using aMicrocaster™ device (Microcaster™, Schleicher & Schüll)768 amplified cDNAs were stamped to a Castslide™

(Schleicher& Schüll) having an area of about 2 cm². Afterthe punctual application of cDNAs the slides were trea-ted with denaturing solution (0.4 M NaOH, 3 × SSC,10 mM EDTA) for 5 minutes and then with a neutraliz-ing buffer (0.5M Tris-HCL pH 7.0, 1.5 M NaCl). For thedifferential hybridization a single stranded cDNA probefrom total RNA of Cuscuta reflexa shoot material with-out prehaustoria and haustoria or with prehaustoriaand haustoria was labelled using Label Star Array Kit(Quiagen) and ³³P.dCTP. After incubation for 1 h at42 °C with PreHyb/Wash Buffer (CAST™ MicroHybridi-zation Kit, Schleicher & Schüll) the slides were incubatedin a volume of 1 ml and 1 million cpm labelled cDNAover night at 42 °C. The slides were washed 3 times for30 minutes at room temperature with PreHyb/Wash Buf-fer and the hybridization signals detected and quantifiedwith a Phoshorimager (BAS-1800 Scanner, BasReader,Fuji). 7000 cDNAs were screened and 16 corresponding

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genes that were identified to be clearly upregulated in thehaustoria containing fraction were identified.Sequence comparison and predictionsSequence based comparisons with the Cuscutain cDNAwere performed by web-based tools at NCBI (http://www.ncbi.nlm.nih.gov). Cleavage site prediction of thededuced Cuscutain pre-pro-protein was determined byapplication of Protein Machine software available atExpasy (http://us.expasy.org/tools/).

Expression and isolation of the propeptide and cuscutainThe plasmid for the expression of the propeptide inhibi-tor fragment (amino acid residues 32-134) and the cuscu-tain enzymatic region (amino acid residues 135-367)were constructed using the GATEWAY™-system (Invitro-gen) according to the manufacturer’s protocol [43]. Entryvector pDonr201 and destination vector pETDest42(Invitrogen) were used for cloning the respective Cuscu-tain cDNA or cDNA fragment in frame with a 6 x Histag at the protein’s C-terminus. The propeptide contain-ing polypeptide and that with cuscutain were expressedin E. coli (BL21Lys). 100 ml of E. coli suspension wasgrown in an appropriate Erlenmeyer flask in LB [44] at37 °C. At an O.D. of 0.8, the 1 mM IPTG was added for 4hours and the suspension centrifuged for 10 min at13.000 rpm. The supernatant was discarded and the cellsresuspended in 4 ml of resuspension buffer (50 MNa-phosphate, pH 7.5, 4 M urea, 300 mM NaCl). TheHIS-tagged propeptide was allowed to bind to pre-equili-brated BD TALON™ metal affinity resin (Clontech) over-night at 4 °C. Subsequently, the resin was washed 3 timeswith resuspension buffer. Bound protein was eluted using1 ml of elution buffer (50 M Na-phosphate, pH 7.5, 4 Murea, 300 mM NaCl and 150 mM Imidazole). Prior tofurther analysis, the obtained solution was dialysedagainst 50 mM Na-phosphate, pH 7.5. E. coli expressingcuscutain were resuspended in 1.5 ml electrophoresisbuffer (0.25 M Tris-HCl, pH6.8, Glycerol 50%, SDS 0.2g), subjected to sonification and separated by SDS-gelelectrophoresis using standard methods [45]. By compar-ison to a parallel Coomassie stained gel, the lanes con-taining cuscutain were identified and cut out. The HIStagged cuscutain enzymatic region was eluted using theElutrap system (Schleicher & Schüll) following to themanufactures’ protocol. Elution was performed overnightat 80 V. SDS was removed from the sample using themethod of Henderson et al. [46]. To ensure cuscutainenzymatic a buffer containing 40 mM Tris/borate, pH8.5, 50% glycerol, 3 mM glutathione was added drop wiseto a sample dilution of 200 : 1 and incubated overnight at4 °C according a protocol provided by Tobbell et al [47].For protein concentration, the solution was electro elutedagain at 200 V. Purified proteins were stored at -20°C.

Kinetic MeasurementsCuscutain activity was determined using the colori-metric papain substrate Cbz-Phe-Arg-pNA. In brief12.08 μM cuscutain and 0.4 mM dipeptide in 500 μl 0.1M Na-citrate, pH 7.5, containing 20% ethanol, wereincubated at 37°C for 10 min. After addition of 500 μl 5mM PMSF in DMSO, absorbance of para-nitroanilinewas measured at a wavelength of 405 nm. Cbz-Phe-Arg-pNA concentrations of 0.2 mM and 0.4 mM were usedfor 1/v versus [I] plots [48]. All measurements for cus-cutain were performed at 30 °C, and assay conditionswere 50 mM Tris-buffer, pH 7.5 containing 300 mMNaCl. The presented data rely on three independentexperiments throughout.

AcknowledgementsThe project was funded by DFG (Deutsche Forschungsgemeinschaft). Wethank David T. Hanson (University of New Mexico) for critical reviewing themanuscript.

Author details1Pathology of Forest Trees, TU Munich, Am Hochanger 13, 85354 Freising,Germany. 2ZMBP, Forschungsgruppe Pflanzenbiochemie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 5, D-72076 Tübingen, Germany.3University of Applied Sciences, Chemistry and Biotechnology,Schnittspahnstr. 12, 64287 Darmstadt, Germany. 4Darmstadt University ofTechnology, Applied Plant Science, Schnittspahnstr. 10, 64287 Darmstadt,Germany.

Author’s contributionsMB: Isolated and characterized cuscutain. Performed biological significancetests. MA: Constructed cDNA-library and screening. LF: Significantlycontributed to enzymatic characterization of cuscutain. RK: Contributed toconception and design, analysis and interpretation of data. Was involved indrafting the manuscript and revising it critically for important intellectualcontent. All authors read and approved the final manuscript.

Received: 19 May 2010 Accepted: 22 October 2010Published: 22 October 2010

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doi:10.1186/1471-2229-10-227Cite this article as: Bleischwitz et al.: Significance of Cuscutain, acysteine protease from Cuscuta reflexa, in host-parasite interactions.BMC Plant Biology 2010 10:227.

Bleischwitz et al. BMC Plant Biology 2010, 10:227http://www.biomedcentral.com/1471-2229/10/227

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